Viroporins in the Influenza Virus
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cells Review Viroporins in the Influenza Virus Janet To and Jaume Torres * School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore * Correspondence: [email protected]; Tel.: +65-6316-2857 Received: 19 May 2019; Accepted: 27 June 2019; Published: 29 June 2019 Abstract: Influenza is a highly contagious virus that causes seasonal epidemics and unpredictable pandemics. Four influenza virus types have been identified to date: A, B, C and D, with only A–C known to infect humans. Influenza A and B viruses are responsible for seasonal influenza epidemics in humans and are responsible for up to a billion flu infections annually. The M2 protein is present in all influenza types and belongs to the class of viroporins, i.e., small proteins that form ion channels that increase membrane permeability in virus-infected cells. In influenza A and B, AM2 and BM2 are predominantly proton channels, although they also show some permeability to monovalent cations. By contrast, M2 proteins in influenza C and D, CM2 and DM2, appear to be especially selective for chloride ions, with possibly some permeability to protons. These differences point to different biological roles for M2 in types A and B versus C and D, which is also reflected in their sequences. AM2 is by far the best characterized viroporin, where mechanistic details and rationale of its acid activation, proton selectivity, unidirectionality, and relative low conductance are beginning to be understood. The present review summarizes the biochemical and structural aspects of influenza viroporins and discusses the most relevant aspects of function, inhibition, and interaction with the host. Keywords: influenza virus; matrix protein 2 (M2); viroporins; ion channel inhibition; protein–protein interactions 1. Introduction 1.1. Influenza Viruses Influenza viruses are enveloped, segmented, negative-sense RNA viruses belonging to the Orthomyxoviridae family. Four influenza virus types have been identified to date, classified based on their core proteins: A, B, C [1] and D [2,3]. Type A is known to infect humans, as well as porcine, bovine and canine species [4]. The main reservoir of the influenza A viruses are aquatic birds [5]. The seasonal flu caused by influenza A virus (IAV) in humans is a very contagious respiratory illness that is among the top ten leading causes of death in the United States and associated with high medical burden [6–8]. Sudden pandemics with high mortality rates are caused by the sporadic transmission of avian or swan influenza viruses to humans, as pre-immunity to these new strains is non-existent [6,7]. Influenza B virus (IBV), its close relative, is less severe but still capable of causing serious outbreaks, is responsible for seasonal influenza epidemics among humans [9,10], and accounts for half of the influenza diseases in recent years (www.cdc.gov). Types B (IBV) and C (ICV) infect humans and pigs [11], whereas type D (IDV) infects cattle and pigs [3]. In addition, IBV has also been reported to infect harbor seals [12]. Although IDV infection in cattle is usually asymptomatic, it can lead to disease in swine [2,13]. Cells 2019, 8, 654; doi:10.3390/cells8070654 www.mdpi.com/journal/cells Cells 2019, 8, 654 2 of 20 1.2. The Proteins in Influenza Viruses In IAV, the viral envelope contains cholesterol-enriched lipid rafts and transmembrane glycoproteins hemagglutinin (HA) and neuraminidase (NA) [14], which are used to further classify IAV strains. A third membrane protein is matrix protein 2 (AM2 in IAV), present in lower abundance [15]. HA, NA, and M2 are type I, type II, and type III membrane proteins, respectively [16–18]. A layer of matrix protein 1 (M1) just underneath the membrane is the most abundant protein of the virus. M1 forms an internal coat, enclosing the viral ribonucleoproteins as well as three polymerase proteins (PA, PB1, and PB2) that form the viral RNA polymerase complex [19]. IAV and IBV have eight negative-sense RNA segments, each encoding one or two viral proteins. IAV encodes 10–11 proteins: HA, NA, NP, M1, M2, PA, PB1, PB2, NS1, NS2, and PB1-F2 [19]. IBV has the same proteins as IAV, except for the PB1-F2 protein, and has an additional surface glycoprotein NB that is unique to flu B. ICV and IDV consist of only seven gene segments each, and they do not encode envelope glycoproteins HA and NA. Instead, they carry only one glycoprotein, HEF (haemagglutinin–esterase fusion), which combines the functions of HA and NA [20]. M2, PB1-F2, and NB proteins are classified as viroporins, i.e., virus-encoded short polypeptides (approximately 60–120 amino acids) that have one, two, or even three [21] transmembrane (TM) α-helical domains. These polypeptides form oligomers of typically four to six monomers that permeabilize membranes to ions [22–24]. 2. AM2 and BM2 2.1. AM2 The RNA segment 7 from IAV encodes both the M1 protein and AM2 proton channel via alternative splicing [25,26]. AM2 is 97-residues long and forms a channel assembled by the tetramerization of four left-handed TM helices stabilized by disulfide bridges [18,27]. The AM2 monomer consists of an N-terminal ectodomain (residues 1–24), an α-helical (TM) domain (residues 25–46), and a C-terminal cytoplasmic tail (CT) domain (residues 47–97) that contains a highly conserved amphipathic helix (APH) (residues 51–59) [25,28]. AM2 forms an acid-activated proton channel with an asymmetrical preference of proton conduction from the viral exterior (N-terminus) to the interior (C-terminus). The proton channel activity of AM2 is essential for two important roles. Upon viral entry into cells via sialic acid-containing receptor-mediated endocytosis, the low pH (5 to 6) of late endosomes activates the proton channel activity of AM2, which acidifies the viral interior [25] with subsequent detachment of M1 from the vRNP complex. This enables trafficking of vRNPs into the host cell nucleus for synthesis of mRNA and vRNA [29,30]. The channel activity of M2 is also crucial during viral maturation, where it equilibrates the pH across the trans-Golgi membrane to prevent the viral HA from undergoing premature conformational change while being transported to the plasma membrane of infected cells [31–34]. Conductance and inhibition studies have used the transmembrane domain of AM2 (AM2-TM), since in Xenopus oocytes AM2(21–51) has retained the proton selectivity and drug sensitivity observed in full-length AM2 protein [35]. Four main features characterize AM2-facilitated membrane permeabilization to protons: acid activation, proton selectivity, relative low proton conductance, and unidirectionality. These features are encoded in key pore-lining residues of the AM2 channel. AM2 has a conserved HxxxW functional motif in its TM domain (Figure1), where His37 is responsible for proton selectivity and acid activation [ 36], and Trp41 ensures asymmetric proton conduction from the N-terminus to the C-terminus [37]. Other residues surrounding this motif also contribute to the dynamics and proton transfer equilibria in the channel. Cells 2019, 8, 654 3 of 20 Cells 2019, 8, x FOR PEER REVIEW 3 of 19 10 20 AM2 1 ...................................MSLLTEVETPIRNEWGCRCN BM2 1 ...................................... ..............ML CM2 1 .............................CNLKTCLKLFNNTDAVTVHCFNENQG DM2 1 MANLALKRSVLTLLMLVICGIPTCVNAETVEEFCRKKLNQTEEKVYVHCFNEDDG 30 40 50 60 70 AM2 21 DSSDPLVVAASIIGI LHLILWILDR LLFFKCIYRFFEHGLKRGPSTEGVPESMREE BM2 3 EPFQILSICSFILSA LHFIAWTIGH LLNQIKRGINMKIRIKGPNKETINREVSILR CM2 27 YMLTLASLGLGIITM LYLLVKIIIE LLVNGFVLGRWERWCGDIKTTIMPEIDSMEK DM2 56 RAMTLAALILGCFSM LYILIKAILM LLLLTIINGRPNGSWDDLKHVVKCFSETGSE 80 90 AM2 76 YRKEQQSAVDADDSHFVSIELE.............................. BM2 58 HSYQKEIQAKETMKEVLSNNMEILSDHIIIEGLSAEEIIKMGETVLEIEELH CM2 82 DIALSRERLDLGEDAPDETDNSPIPFSNDGIFEI.................. DM2 111 NFARDIMVLESRRDGEETSSPEEGLGPPLSGFNENGVFMETL.......... FigureFigure 1. SequenceSequence alignmentalignment of of M2 M2 viroporins viroporins in influenza. in influenza. Influenza Influenza A M2 (AM2;A M2 strain (AM2; A/ Udornstrain/ 1972 A/Udorn/1972H3N2), influenza H3N2), B M2influenza (BM2; B strain M2 (BM2; B/Taiwan strain/70061 B/Taiwan/70061/2006),/2006), influenza Cinfluenza M2 (CM2; C M2 strain (CM2; C/ Ann strainArbor C/Ann/1/1950) Arbor/1/1950) and influenza andD influenza M2 (DM2; D M2 strain (DM2; D/ swinestrain /D/swine/Oklahoma/1334/2011).Oklahoma/1334/2011). The predicted The predictedtransmembrane transmembrane regions areregions underlined. are underlined. The functional The functional motifs HxxxWmotifs HxxxW (in AM2 (in and AM2 BM2) and and BM2) YxxxK and(in CM2YxxxK and (in DM2) CM2 areand indicated DM2) are in indicated bold red font.in bo Numberingld red font. Numbering corresponds corresponds to AM2. Sequences to AM2. were Sequencesretrieved fromwere retrieved UniProt ( www.uniprot.orgfrom UniProt (www.uniprot.org). ). 2.2.2.2. BM2 BM2 InIn IBV, IBV,RNA RNA segmentsegment 77 encodes encodes both both M1 M1 protein protei andn and BM2 BM2 [38 [38,39].,39]. Like Like AM2, AM2, BM2 BM2 is a pH-activatedis a pH- activatedproton channel proton channel [40] and [40] has and a similar has a similar monomeric monomeric and oligomericand oligomeric organization organization as describedas described above abovefor AM2 for [AM241,42 ].[41,42]. Like AM2,Like AM2, a truncated a truncated peptide peptide containing containing its TM, its BM2(1–33),TM, BM2(1–33), conducts conducts protons protonswhen incorporated when incorporated into artificial into liposomesartificial liposomes [42] and Xenopus